- Cutting inserts to be placed in mammalian vector
A) Get biobricks and enzymes from freezer.
The following list of parts are:
Follow steps for gel electrophoresis.
- Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
- Fill gel flask with up to 60 ml of TA buffer.
- Create 1% gel by putting .6 grams of agarose into flask.
- Microwave agarose solution for 40 seconds
- Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
- When flask is taken out of microwave, make sure that the agarose is completed dissolved.
- Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches.
- Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
- Pour gel into tray.
- Wash the agarose gel flask.
- Run gel for 45 min at 100 V. Check gel under UV light.
30 μL/lane, 1% agarose; Ladder
| DNA (plasmid) || 20.0 μL
| SpeI|| 1.0 μL
| PstI|| 1.0 μL
| Green 10x FastDigest buffer || 3.0 μL
| dH2O || 5 μL
| || 30 μL --> 37°C/ ~10 min.